Cephalosporin compositions and methods of manufacture

ABSTRACT

Provided herein is a method for the synthesis of cephalosporin antibiotic compounds comprising the conversion of a protected 7-amino group into a 7-carboxamide moiety in a single step.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 61/782,365, filed Mar. 14, 2013. The entire content of this application is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to cephalosporin compositions and the manufacture thereof.

BACKGROUND

Cephalosporin compounds containing the chemical substructure of formula (I) are important antibacterial therapeutic agents. The manufacture of these cephalosporin compounds is typically performed in a series of synthetic chemical reactions. Reducing the number of synthetic chemical reactions and associated purification steps can increase product yield and decrease the time for production, thereby increasing efficiency and decreasing production costs.

Syntheses of antibiotic cephalosporin compounds containing the substructure of formula (I) typically require two reactions for the formation of a 7-carboxamide moiety: (1) deprotection (i.e., removal of a protecting group) of the 7-amino group, and (2) acylation of the 7-amino group to form the 7-carboxamide moiety. One example of this two-step procedure is disclosed in U.S. Pat. No. 5,134,138 (see FIG. 2). Most cephalosporin antibiotics can be manufactured in an analogous way. Ceftolozane (CXA-101, FR264205) is a cephalosporin antibiotic compound, a synthesis of which is disclosed in U.S. Pat. No. 7,129,232. Additional cephalosporin antibiotic compounds include the compounds of Table 2. There remains an unmet need to identify novel manufacturing processes for synthesizing cephalosporin compounds comprising the chemical substructure of formula (I), including methods for formation of a 7-carboxamide moiety from a protected 7-amino group with fewer chemical synthetic steps.

SUMMARY

A novel chemical process useful in the manufacture of cephalosporin compounds containing the chemical substructure of Formula (I) can include deprotection (i.e., removal of a nitrogen protecting group) and acylation of the 7-amino group in a single step rather than multiple steps. The invention is based in part on the surprising discovery that salicyladehyde imine derivatives according to formula (Ia) can be reacted with activated carboxylic acid derivatives (Ib) to yield 7-carboxamide compounds according to formula (I) in a single step. This outcome is surprising because the imine derivatives of formula (Ia) are stable to the reaction conditions in the absence of the activated carboxylic acid derivative.

Accordingly, provided herein is a method for transforming a protected 7-amino group into a 7-carboxamide moiety comprising a single step.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a representative 7-carboxamide moiety and 7-amino group.

FIG. 2 depicts a prior art procedure (U.S. Pat. No. 5,134,138) comprising the deprotection of a protected 7-amino group followed by conversion of the 7-amino group into a 7-carboxamide moiety.

FIG. 3 depicts the full scan mass spectrum of compound (IV-1).

FIG. 4 depicts the product ion mass spectrum of compound (IV-1).

FIG. 5 depicts the full scan mass spectrum of compound (III-1).

FIG. 6 depicts the product ion mass spectrum of compound (III-1).

FIG. 7 depicts the full scan mass spectrum of compound (II-1).

FIG. 8 depicts the product ion mass spectrum of compound (II-1).

FIG. 9 depicts the full scan mass spectrum of ceftolozane trifluoroacetate.

FIG. 10 depicts the product ion mass spectrum of ceftolozane trifluoroacetate.

FIG. 11 depicts the HPLC chromatograms of Example 2.

DETAILED DESCRIPTION

Provided herein is a method for preparing a compound of formula (II), or a salt thereof, comprising the step of reacting a compound of formula (III), or a salt thereof, with a compound of formula (IV), or a salt thereof, under suitable conditions to form a compound of formula (II), or a salt thereof, wherein the compounds of formulas (II), (III) and (IV) are defined according to the variable values below.

Variable R₁

In certain embodiments, R₁ is R_(1′)—Z; wherein R_(1′) is selected from the group consisting of a bond, aryl, cycloalkyl, cycloalkenyl, heterocyclyl, and heteroaryl; wherein Z is 0-2 instances of a substituent that for each occurrence is independently selected from the group consisting of hydrogen, halogen, hydroxyl, hydroxyalkyl, aminoalkyl, alkyl, alkylidenyl, alkyenyl, heteroalkyl, cyano and amino, wherein Z is optionally, independently substituted one or more times with amino, halogen, carboxyl, oxo, a nitrogen protecting group, an oxygen protecting group or —P(O)(OZ′)₂; and wherein Z′ is independently hydrogen or an oxygen protecting group.

In one embodiment, R₁ is selected from the group consisting of aryl and heteroaryl moieties. In certain embodiments, R₁ is a substituted or unsubstituted aryl or heteroaryl moiety selected from the group consisting of: thiophene, furan, thiazole, tetrazole, thiadiazole, pyridyl, phenyl, phenol, cyclohexadiene and dithietane. In a particular embodiment, R₁ is selected from the group consisting of the following moieties:

and salts thereof, wherein Z′ is defined above. In another particular embodiment, R₁ is

and the compound of formula (II) has the structure of formula (Ila).

Variable R₂

In certain embodiments, R₂ is selected from the group consisting of hydrogen and alkoxy. In another embodiment of the method, R₂ is hydrogen, and the compound of formula (II) has the structure of formula (IIb).

Variable Y

In certain embodiments, Y is selected from the group consisting of a bond, CH₂, CH₂S, SCH₂, C≡C(H)CH₂CO₂R′, CH(OR′), C≡N(OR′), CHNR″₂ and C≡NR″; wherein R′ is selected from the group consisting of hydrogen, an oxygen protecting group and alkyl, wherein the alkyl is optionally substituted one or more times with halogen, hydroxyl or —CO₂R₅; wherein R″ is a substituent that for each occurrence is selected from hydrogen, alkyl, C(O)heterocyclyl, and a nitrogen protecting group, wherein any two R″ substituents may combine to form a ring or a single nitrogen protecting group; and wherein R₅ is independently hydrogen or an oxygen protecting group.

In one embodiment of the method, Y is selected from a bond, CH₂, CH₂S and C≡C(H)CH₂CO₂R′, and R′ is selected from the group consisting of hydrogen and an oxygen protecting group. In another embodiment, Y is C≡N(OR′) and R′ is selected from the group consisting of an oxygen protecting group, hydrogen, methyl, ethyl, CH₂CO₂R₅ and C(CH₃)₂CO₂R₅. In yet another embodiment, Y is CH(OR′) and R′ is hydrogen or an oxygen protecting group. In still another embodiment, Y is CHNR″₂ and R″ is 1-2 instances of a substituent that for each occurrence is selected from hydrogen, a nitrogen protecting group and

In a particular embodiment, Y is C≡N(OR′) and R′ is C(CH₃)₂CO₂ ^(t)Bu, and the compound of formula (II) has the structure of formula (IIc).

Variable R₃

In certain embodiments, R₃ is selected from the group consisting of hydrogen and an oxygen protecting group. In another embodiment of the method, R₃ is an oxygen protecting group selected from benzyl ethers. Benzyl ethers may be substituted (e.g., with one or more alkoxy substituents) or unsubstituted. In a particular embodiment, R₃ is

(i.e., 4-methoxybenzyl, PMB, MPM).

Variables R₄ and R_(4′)

R₄ and R_(4′), are independently selected from the group consisting of halogen, —R₆-R₇, —CH₂—R₆-R₇ and —CH═R₇; wherein R₆ is selected from the group consisting of a bond, oxygen, sulfur, alkyl, alkenyl, aryl, heteroaryl and heterocyclyl; wherein R₇ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkanoyl, aroyl, heteroaroyl, carboxamide, aryl, heteroaryl and heterocyclyl; and wherein R₇ is optionally, independently substituted 1-3 times with a substituent selected from alkyl, alkyl sulfite, heterocyclyl and NR_(a)R_(b); wherein R_(a) and R_(b) are independently selected from the group consisting of hydrogen, alkyl, a nitrogen protecting group and C(O)NHR_(c); wherein R_(c) is an alkyl group or a heteroalkyl group; and wherein R₄ and R_(4′) are optionally, independently substituted one or more times with an oxygen protecting group or a nitrogen protecting group.

In another embodiment of the method, R₄ and R_(4′), are independently selected from the group consisting of hydrogen, chlorine, methyl, CH═CH—CH₃, CH═CH₂, CH₂OC(O)CH₃, CH₂OC(O)NH₂, CH₂OCH₃,

wherein A^(⊖), for each occurrence, is independently a pharmaceutically acceptable anion. In certain embodiments, A^(⊖) is selected from chloride, acetate, trifluoroacetate and bisulfate. In a particular embodiment, A^(⊖) is trifluoroacetate.

In a particular embodiment, R₄ is

In another particular embodiment, R_(4′), is

Variable X

In certain embodiments, X is selected from the group consisting of halogen, —OC(O)R₈ and —OSO₂R₈; wherein R₈ is selected from the group consisting of alkyl, heteroalkyl, aryl and heteroaryl. In another embodiment of the method, X is —OSO₂R₈. In a particular embodiment, X is —OSO₂CH₃.

(II-1), (III-1) and (IV-1)

Particular embodiments of the compounds of formulas (II), (III) and (IV) are listed in Table 1. For example, in the synthesis of the antibiotic compound ceftolozane, the compounds of formulas (II), (III) and (IV) can have the structures of formulas (II-1), (III-1) and (IV-1), respectively.

Synthesis

In one embodiment of the method, the suitable conditions comprise reacting in a mixture comprising an organic solvent and water. Organic solvents can be selected from the group consisting of aromatic solvents, aliphatic solvents, halogenated solvents, halogenated aromatic solvents, halogenated aliphatic solvents, ethers, halogenated ethers, esters and amides. In a particular embodiment, the organic solvent is selected from halogenated solvents. In another particular embodiment, the organic solvent is dichloromethane.

In another embodiment of the method, the suitable conditions comprise reacting in a mixture comprising an organic solvent and about 6-9 percent (w/w) water relative to the compound of formula (III), or salt thereof. In a particular embodiment, the suitable conditions comprise reaction in a mixture comprising an organic solvent and about 6-8 percent (w/w) water relative to the compound of formula (III), or salt thereof. In still another embodiment, the suitable conditions comprise reacting in a mixture comprising dichloromethane and about 6-9 percent of water relative to the compound of formula (III), or salt thereof.

Notably, the compound of formula (III) is hydrolytically stable under the reaction conditions, in the absence of the compound of formula (IV). As shown in FIG. 11 and described in Example 2, compound (III-1) was combined with dichloromethane and water in the same relative amounts as in Example 1. Upon stirring for four hours, no hydrolysis of (III-1) was observed. In contrast, under the same solvent, temperature and time conditions, but in the presence of compound (IV-1), compound (III-1) reacts to form compound (II-1).

In some embodiments, the method further comprises the step of reacting the compound of formula (II), or a salt thereof, under suitable conditions to form a compound of formula (V), or a salt thereof, wherein the compound of formula (V) is selected from the compounds of Table 2, or salts thereof. In some embodiments, the suitable conditions comprise reacting the compound of formula (II) with strong acid. In a particular embodiment, the strong acid is trifluoroacetic acid.

Compositions

In another aspect, provided herein is a composition comprising the compound of formula (V), or salt a thereof, and one or more compound selected from the group consisting of: the compound of formula (II), or salt a thereof, the compound of formula (III), or salt a thereof, and the compound of formula (IV), or salt a thereof. In one embodiment, the composition comprises the compound of formula (V), or salt a thereof, the compound of formula (I) or salt a thereof, the compound of formula (Ia) or salt a thereof, and the compound of formula (Ib) or salt a thereof. In another embodiment of the composition, the compound of formula (II), or salt a thereof, the compound of formula (III), or salt a thereof, and the compound of formula (IV), or salt a thereof, are present, if at all, in an amount that is less than or equal to 0.05% w/w. In yet another embodiment of the composition, the compound of formula (II), or salt a thereof, the compound of formula (III), or salt a thereof, and the compound of formula (IV), or salt a thereof, are present, if at all, in an amount that is less than or equal to 0.05 mol %. In another aspect, provided herein is a method of treating a bacterial infection in a patient, comprising the step of administering a composition as described above.

DEFINITIONS

Pharmaceutically acceptable salts are known to those of skill in the art. In some of the embodiments described herein, the compounds of formulas (II) and (III) are trifluoroacetate salts.

Oxygen and nitrogen protecting groups are known to those of skill in the art. Oxygen protecting groups include, but are not limited to methyl ethers, substituted methyl ethers (e.g., MOM (methoxymethyl ether), MTM (methylthiomethyl ether), BOM (benzyloxymethyl ether), PMBM or MPM (p-methoxybenzyloxymethyl ether), to name a few), substituted ethyl ethers, substituted benzyl ethers, silyl ethers (e.g., TMS (trimethylsilyl ether), TES (triethylsilylether), TIPS (triisopropylsilyl ether), TBDMS (t-butyldimethylsilyl ether), tribenzyl silyl ether, TBDPS (t-butyldiphenyl silyl ether), to name a few), esters (e.g., formate, acetate, benzoate (Bz), trifluoroacetate, dichloroacetate, to name a few), carbonates, cyclic acetals and ketals. Nitrogen protecting groups include, but are not limited to, carbamates (including methyl, ethyl and substituted ethyl carbamates (e.g., Troc), to name a few) amides, cyclic imide derivatives, N-alkyl and N-aryl amines, benzyl amines, substituted benzyl amines, trityl amines, imine derivatives, and enamine derivatives, for example.

In some embodiments, alkyl groups and groups comprising an alkyl group (e.g., alkoxy, alkanoyl, alkylamino, aminoalkyl, heteroalkyl) comprise 1-6 carbon atoms. In some embodiments, aryl groups and groups comprising aryl groups (e.g., aroyl) comprise 6-12 carbon atoms. In some embodiments, heteroaryl groups and groups comprising heteroaryl groups (e.g., heteroaroyl) comprise 1-10 carbon atoms and 1-4 heteroatoms selected from oxygen, nitrogen and sulfur.

TABLE 1 Formula (II) Formula (III) Formula (IV)  1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

TABLE 2 Formula (V) V-1

V-2

V-3

V-4

V-5

V-6

V-7

V-8

V-9

V-10

V-11

V-12

V-13

V-14

V-15

V-16

V-17

V-18

V-19

V-20

V-21

V-22

V-23

V-24

V-25

V-26

V-27

V-28

V-29

V-30

V-31

V-32

EXAMPLES Materials and Instrumentation

Compound characterization was obtained by high resolution mass spectrometry (HRMS) and high resolution mass spectrometry/mass spectrometry (HR MS/MS). Samples were dissolved in solution and the sample solutions were directly infused into time-of-flight (TOF) mass spectrometer to obtain parent (MS) and product scans (MS/MS) of analyte. Suitable materials and instrumentation are known to persons of skill in the art.

Example 1 Synthesis of Compound (II-1) (1) Synthesis of compound (IV-1): (Z)-2-(5-amino-1,2,4-thiadiazol-3-O-2-(((1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl)oxy)imino)acetic methanesulfonic anhydride

A 5.0 liter reactor was charged with N,N-dimethylacetamide (1.4 L). Solid (Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(((1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl)oxy)imino)acetic acid (250 g, 1 eq., prepared according to the method disclosed in U.S. Pat. No. 7,129,232) was added to the reactor at ambient temperature and the mixture was stirred until the solid was dissolved. The solution was cooled to about 4° C. (target: 2-5° C.) and stirred for another 10-15 minutes. Potassium carbonate (106.7 g, 184.9 mmol, 1.1 eq.) was added to the reactor in one portion. Methanesulfonyl chloride (118.0 mL, 2.2 eq.) was added to the reactor at a rate of about 5 mL/min while maintaining the batch temperature <10° C. The reaction was stirred for 1-2 hours at about 6° C., then cooled to about 0-5° C. Ethyl acetate (2.5 L, 10 volumes) was added to the reactor while maintaining a temperature of about 0-5° C. 1.6% HCl (aq) (1.35 L) was added to the reactor, maintaining a temperature of about 10-15° C. The biphasic mixture was agitated and the layers were then separated. The organic layer was washed with sodium chloride solution, dried with sodium sulfate and concentrated under reduced pressure to obtain crude material. The crude material was dissolved in ethyl acetate (150 mL) and filtered over silica gel. The filtrate was concentrated under reduced pressure to yield the title compound (299 g, 96.7%) as a colorless solid. Exact Mass: 408.08. HRMS: 409.0846 (M+1).

(2) Synthesis of compound (III-1): 5-amino-4-(3-(2-((tert-butoxycarbonyl)amino)ethyl)ureido)-2-(((6R,7R)-7-((Z)-(2-hydroxybenzylidene)amino)-2-(((4-methoxybenzyl)oxy)carbonyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl)methyl)-1-methyl-1H-pyrazol-2-ium trifluoroacetate

Compound (III-1) was prepared by coupling the compound (6R,7R)-4-methoxybenzyl 3-(chloromethyl)-7-((Z)-(2-hydroxybenzylidene)amino)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate (purchased from Nippon Chemicals, Japan) with the compound tert-butyl (2-(3-(1-methyl-5-(tritylamino)-1H-pyrazol-4-yl)ureido)ethyl)carbamate (prepared according to the method disclosed in U.S. Pat. No. 7,129,232). Exact Mass (-TFA): 977.40. HRMS: 977.4025 (M+).

(3) Synthesis of compound (II-1): 5-amino-2-(((6R,7R)-7-((Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(((1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl)oxy)imino)acetamido)-2-(((4-methoxybenzyl)oxy)carbonyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl)methyl)-4-(3-(2-((tert-butoxycarbonyl)amino)ethyl)ureido)-1-methyl-1H-pyrazol-2-ium trifluoroacetate

A reactor was charged with compound (III-1) (150 g), water (11.25 mL), compound (IV-1) (60 g), and dichloromethane (265 mL). The mixture was stirred for three hours at room temperature, then added to methyl tertiary butyl ether (1.5 L) over the course of 2-3 hours, resulting in the formation of a solid. The solid was filtered and the filter cake was washed with additional methyl tertiary butyl ether (1.0 L). The solid was dried under vacuum to yield the title compound (142.1 g, 81.2%). Exact Mass: 943.36 (-TFA). HRMS: 943.3555 (M+).

(4) Synthesis of Ceftolozane Trifluoroacetate

Compound (II-1) was dissolved in dichloromethane. Trifluoroacetic acid and anisole were added and the reaction was stirred for about 3 hours. Methyl tertiary butyl ether was added, causing the formation of a precipitate. The precipitate was collected by filtration and dried under vacuum to yield the title compound. Exact Mass (-TFA): 667.18. HRMS: 667.1810 (M+).

Example 2

Compound (III-1) (2.12 g) was dissolved in dichloromethane (3.75 ml). Water (160 uL) was added and the mixture was stirred for four hours, with hourly monitoring by HPLC.

Example 3 Synthesis of 4-(2-W7R)-7-((Z)-2-(ethoxyimino)-2-(5-(phosphonoamino)-1,2,4-thiadiazol-3-yl)acetamido)-2-(((4-methoxybenzyl)oxy)carbonyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl)thio)thiazol-4-yl)-1-methylpyridin-1-ium trifluoroacetate

A reactor is charged with 4-(2-(((7R)-7-((Z)-(2-hydroxybenzylidene)amino)-2-(((4-methoxybenzyl)oxy)carbonyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl)thio)thiazol-4-yl)-1-methylpyridin-1-ium trifluoroacetate (1 eq), water (6-9 weight % relative to the Schiff base), (Z)-2-(5-(phosphonoamino)-1,2,4-thiadiazol-3-yl)-2-(ethoxyimino)acetic methanesulfonic anhydride (2.5 eq) and dichloromethane (1.75 mL per gram of Schiff base). The mixture is stirred for three hours at room temperature, then added to methyl tertiary butyl ether over the course of 2-3 hours, resulting in the formation of a solid. The solid is filtered and the filter cake was washed with additional methyl tertiary butyl ether. The solid is dried under vacuum to yield the title compound.

Example 4 Synthesis of 4-methoxybenzyl 7((Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(((4-methoxybenzyl)oxy)imino)acetamido)-3-(E)-((R)-1′-(tert-butoxycarbonyl)-2-oxo-[1,3′-bipyrrolidin]-3-ylidene)methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

A reactor is charged with (7R)-4-methoxybenzyl-3-((E)-((R)-1′-(tert-butoxycarbonyl)-2-oxo-[1,3′-bipyrrolidin]-3-ylidene)methyl)-7-((Z)-(2-hydroxybenzylidene)amino)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate (1 eq), water (6-9 weight % relative to the Schiff base), (Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(((4-methoxybenzyl)oxy)imino)acetic methanesulfonic anhydride (2.5 eq) and dichloromethane (1.75 mL per gram of Schiff base). The mixture is stirred for three hours at room temperature, then added to methyl tertiary butyl ether over the course of 2-3 hours, resulting in the formation of a solid. The solid is filtered and the filter cake was washed with additional methyl tertiary butyl ether. The solid is dried under vacuum to yield the title compound.

Example 5 Synthesis of 1-W6R,7R)-7-((Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(((1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl)oxy)imino)acetamido)-2-(((4-methoxybenzyl)oxy)carbonyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl)methyl)pyridin-1-ium trifluoroacetate

A reactor is charged with 1-(((7R)-7-((Z)-(2-hydroxybenzylidene)amino)-2-(((4-methoxybenzyl)oxy)carbonyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl)methyl)pyridin-1-ium trifluoroacetate (1 eq), water (6-9 weight % relative to the Schiff base), (Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(((1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl)oxy)imino)acetic methanesulfonic anhydride (2.5 eq) and dichloromethane (1.75 mL per gram of Schiff base). The mixture is stirred for three hours at room temperature, then added to methyl tertiary butyl ether over the course of 2-3 hours, resulting in the formation of a solid. The solid is filtered and the filter cake was washed with additional methyl tertiary butyl ether. The solid is dried under vacuum to yield the title compound.

Example 6 Synthesis of (7R)-4-methoxybenzyl 74(R)-2-((tert-butoxycarbonyl)amino)-2-phenylacetamido)-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

A reactor is charged with (7R)-4-methoxybenzyl 7-((Z)-(2-hydroxybenzylidene)amino)-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate (1 eq), water (6-9 weight % relative to the Schiff base), (R)-2-((tert-butoxycarbonyl)amino)-2-phenylacetic methanesulfonic anhydride (2.5 eq) and dichloromethane (1.75 mL per gram of Schiff base). The mixture is stirred for three hours at room temperature, then added to methyl tertiary butyl ether over the course of 2-3 hours, resulting in the formation of a solid. The solid is filtered and the filter cake was washed with additional methyl tertiary butyl ether. The solid is dried under vacuum to yield the title compound.

Example 7 Synthesis of 4-methoxybenzyl 3-((carbamoyloxy)methyl)-7-methoxy-8-oxo-7-(2-(thiophen-2-yl)acetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

A reactor is charged with (75)-4-methoxybenzyl 3-((carbamoyloxy)methyl)-7-((Z)-(2-hydroxybenzylidene)amino)-7-methoxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate (1 eq), water (6-9 weight % relative to the Schiff base), 2-(thiophen-2-yl)acetic methanesulfonic anhydride (2.5 eq) and dichloromethane (1.75 mL per gram of Schiff base). The mixture is stirred for three hours at room temperature, then added to methyl tertiary butyl ether over the course of 2-3 hours, resulting in the formation of a solid. The solid is filtered and the filter cake was washed with additional methyl tertiary butyl ether. The solid is dried under vacuum to yield the title compound. 

1. A method for preparing a compound of formula (II-1), or a salt thereof,

comprising the step of reacting a compound of formula (III-1), or a salt thereof,

with a compound of formula (IV-1), or a salt thereof,

in a mixture comprising an organic solvent and about 6-9 percent (w/w) of water relative to the compound of formula (III-1), or salt thereof.
 2. The method of claim 1, wherein the organic solvent is dichloromethane.
 3. The method of claim 1, wherein the compound of formula (IV-1) is prepared from the compound of formula (IVa):

by a process comprising the steps of: (a) forming a mixture comprising potassium carbonate and the compound of formula (IVa); and (b) adding methansulfonyl chloride to the mixture of step (a) such that the compound of formula (IV-1) is formed.
 4. The method of claim 1, further comprising the step of reacting the compound of formula (II-1), or a salt thereof, with a strong acid to form a compound of formula (V-1), or a salt thereof:


5. The method of claim 4, wherein the strong acid is trifluoroacetic acid.
 6. A composition comprising two or more compounds selected from the group consisting of: (a) the compound of formula (II-1), or a salt thereof

(b) the compound of formula (III-1), or a salt thereof

and (c) the compound of formula (IV-1), or a salt thereof


7. The composition of claim 6, comprising the compound of formula (II-1), or a salt thereof, and the compound of formula (III-1), or a salt thereof.
 8. The composition of claim 7, comprising the compound of formula (II-1), or a salt thereof, the compound of formula (III-1), or a salt thereof, and the compound of formula (IV-1), or a salt thereof.
 9. The composition of claim 6, further comprising the compound of formula (V-1), or salt a thereof


10. The composition of claim 9, wherein the compound of formula (II-1), or salt a thereof, the compound of formula (III-1), or salt a thereof, and the compound of formula (IV-1), or salt a thereof, are each present in an amount that is less than 0.05%.
 11. The composition of claim 9, prepared by a method comprising the steps of: (a) reacting a compound of formula (III-1), or a salt thereof, with a compound of formula (IV-1), or a salt thereof, in a mixture comprising an organic solvent and about 6-9 percent (w/w) of water relative to the compound of formula (III-1), or salt thereof; and (b) reacting the compound of formula (II-1), or a salt thereof, with a strong acid to form a compound of formula (V-1), or a salt thereof.
 12. A compound selected from the group consisting of:


13. The compound of claim 12 having the structure


14. The compound of claim 12 having the structure


15. The method of claim 1, wherein the weight ratio of the compound of formula (III-1) to the compound of formula (IV-1) is 150 g:60 g.
 16. The method of claim 1, wherein the organic solvent is dichloromethane; wherein the compound of formula (IV-1) is prepared from the compound of formula (IVa):

by a process comprising the steps of: (a) forming a mixture comprising potassium carbonate and the compound of formula (IVa); and (b) adding methansulfonyl chloride to the mixture of step (a) such that the compound of formula (IV-1) is formed; wherein the method further comprises the step of reacting the compound of formula (II-1), or a salt thereof, with a strong acid to form a compound of formula (V-1), or a salt thereof:

and wherein the strong acid is trifluoroacetic acid. 